Aqueous composition comprising a biological antigen and an acrylic acid polymer

ABSTRACT

The current invention pertains to an aqueous composition containing a biological antigen and an acrylic acid polymer, wherein the composition comprises an electrolyte to provide an osmolarity higher than the osmolarity of a 0.9% (w/v) sodium chloride solution in water. The invention also pertains to the acrylic acid polymer for use in a one shot vaccine against porcine circo virus 2 (PCV2) and optionally  Mycoplasma hyopneumoniae  and in an aqueous composition for reducing fever induced by the biological antigens present in the aqueous composition when the composition is administered to a subject animal.

The present invention pertains to an aqueous composition comprising abiological antigen and an acrylic acid polymer. The invention alsopertains to the acrylic acid polymer for use in a one shot vaccineagainst porcine circo virus 2 (PCV2) and Mycoplasma hyopneumoniae and inan aqueous composition for reducing fever induced by the biologicalantigens present in the aqueous composition when the composition isadministered to a subject animal.

BACKGROUND ART

It is known for example from WO 2010/025469 to provide an aqueouscomposition (i.e. a composition based on water or another hydrophilicliquid that allows the formation of hydrogen bonds) comprising abiological antigen and an acrylic acid polymer (a biological antigenbeing an antigen derived from a living organism such as e.g. abacterium, virus, animal, protist, fungus etc., typically a live orkilled microorganism, or a biological molecule, preferably a protein orpolysaccharide, derived from the living organism; the term “derivedfrom” encompasses that the biological molecule itself or a precursorthereof is produced by the organism). The acrylic acid polymer in thisprior art reference is used as an adjuvant, i.e a non-specificimmunostimulating agent included in the composition to favor or amplifya particular process in the cascade of immunological events, ultimatelyleading to a better immunological response. Acryclic acid polymers havesince long been recognized as safe and adequate adjuvants, capable ofimproving the immunological response to various types of antigens suchas live or killed microorganisms, subunits of these organisms orrecombinantly produced subunits such as proteins, polysaccharides andother types of molecules. For example, U.S. Pat. No. 3,178,350 alreadydescribes the use of an acrylic acid polymer as an adjuvant. Theseadjuvants are available under the trademark Carbopol™.

A notable property of acrylic acid polymers is that they significantlyincrease the viscosity of an aqueous composition since they inherentlyprovide linked polymer chains in such compositions. The acid residues ofthe polymer chains namely may interact by hydrogen-bonding. Althoughhydrogen bonds are significantly less strong than covalent bonds, thisinteraction between the polymers chains may have a significant influenceon the viscosity of the aqueous solution. This inherent property ofacrylic acid polymers, which property is hardly depended on polymerchain length and type of side chains/groups, is widely used in forexample creams or lotions where acrylic acid polymers are used asthickening agents. When the linked polymers form a true network ofpolymer chains throughout the composition (above a certain polymerconcentration, typically above 0.2 to 0.5% w/w), and the interstices ofthe network are filled with the continuous phase, the composition iscalled a gel. For topical applications of aqueous compositions (such ashand creams, sun tan lotions) this is a preferred state of thecomposition. For compositions containing biological antigens, whichcompositions typically are administered via injection, the increase inviscosity is an important disadvantage. Even when the viscosity of thecomposition rises from about 5-70 mPas (which is typical for an aqueouscomposition comprising antigens) to about 200 mPas, this is verynoticeable when applying the composition by injection (by hand) to asubject animal (the term animal in this specification and appendedclaims includes a human being). The gelling of the composition iscommonly avoided at all times: gelled compositions cannot be readilyinjected. Therefore in practice, for compositions comprising biologicalantigens typically 0.1 to a maximum of 0.2% (weight over weight; w/w) ofan acrylic acid polymer is applied. Indeed, in WO 2010/025469 whichpertains to state-of-the art vaccines, 0.1% w/w of the acrylic acidpolymer is applied. An example of a commercially available vaccinecomprising an acrylic acid polymer is Suvaxyn™ M. Hyo—Parasuis(available from Pfizer Animal Health), which contains 0.2% (w/w)Carbopol™ 941.

It has been described in the art (U.S. Pat. No. 3,920,811) to add aphysiologically acceptable electrolyte (i.e. a compound that ionizeswhen dissolved or molten to produce an electrically conductive medium),such as the electrolytes mentioned in column 4, lines 28-42 of the '811patent) to an injectable composition in order to lower the viscosity ofthe composition to the point where they can be utilized commercially ina practical manner, while at the same time retaining the favorableadjuvant properties. As indicated however clearly in the '811 patent(column 7, lines 3-6), the electrolyte concentration may be as high asthat which produces isotonicity in the final injectable solution of themixture of the adjuvant solution and the biologic antigen. Inparticular, the actual injectable compositions are typically aboutone-half isotonic. The reason for this is that it is generally knownthat hypertonic solutions may provide tissue damage upon injection. Evenwhen the composition is only slightly hypertonic and thus, the ultimatedamage may be neglectable, the result of injection is that the animalmay have a very uncomfortable feeling at the injection site, which mayfor example lead to stress, itching, biting etc. Therefore, in order tocomply with the generally required safety standards for compositions foradministration to animals, each commercially available compositioncomprising biological antigens and an acryl acid polymer, is at mostisotonic with normal body fluid (e.g. serum), i.e. having an osmolarityof a 0.9% (w/v) sodium chloride solution in water (about 300 mOsm/l).

SUMMARY OF THE INVENTION

It is an object of the invention to provide new compositions accordingto the preamble that have wider applicability.

It has been surprisingly found that in a composition comprising an acrylacid polymer, the use of an amount of electrolyte that provides ahypertonic solution still leads to a composition that complies withgenerally accepted safety standards. Apparently, without having a cleartechnical reason for the composition being safe even when it issignificantly hypertonic, the presence of the acrylic acid polymerprovides for a composition wherein the presence of an excess ofelectrolyte does not induce a problem, even upon injection of thecomposition in animal tissue. Without being bound to theory, it isbelieved that the presence of the polymer in the form of a network oflinked polymer chains may constitute a composition wherein a controlledbut fast dilution of the excess electrolyte takes place without damagingthe surrounding tissue. This indeed contradicts the teaching of the '811patent which has been the commonly accepted teaching ever since thepatent was published. One of the major advantages of the present findingthat an excess electrolyte can be used, is that the viscosity of thecomposition comprising the acrylic acid polymer can be furtherdecreased, thereby allowing easier practical utilization of thecomposition. Also, the present invention allows the use of far greateramounts of an acrylic acid polymer, even above 0.5% w/w, while stillretaining a very low viscosity. The use of such amounts of polymer opensthe door to (very) slow-release formulations for the biologic antigens.Given the apparent feature that the electrolyte dilutes fast from thecomposition upon administration into animal tissue, the viscosity of thecomposition will immediately after administration increasesignificantly, and may even form a gelled composition at the site ofadministration. Inherently, the biological antigens will be releasedslower from such a gel when compared to release from an aqueous (lowviscous) composition as such, depending i.a. on the gel properties. Ingeneral, the higher the viscosity, the slower release of antigens is tobe expected.

In particular it has been found that an acrylic acid polymer in anaqueous composition comprising biological antigens can be used forreducing fever induced by these biological antigens when the compositionis administered to a subject animal. Although a temperature rise of 2°C. within the first 24 hours after injection is generally accepted andregarded safe, a temperature rise below 1° C. is generally favourable.With the compositions according to the current invention, a temperaturerise of less the 1° C. or even less then 0.5° C. may be obtained. Thereason for this remains unclear but may be linked to the fact that thehypertonic low viscous solution will almost immediately afteradministration reach a higher viscosity and thus turn into a more orless slow-release formulation. Slow release may result in animmunological response that is less intense, but more prolonged.Although one would expect to see at least some decrease in titre aswell, it was very surprisingly found that still high titres can beobtained, even when the fever is decreased significantly. This opensdoors to obtain safer vaccines based on antigens that typically inducefever such as for example lipopolysaccharide (LPS) containing antigensas present in many vaccines containing gram-negative bacteria such asfor example Escherichia coli (E. coli), Salmonella spp, Shigella spp andother Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter,Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella andalpha-proteobacteria as Wolbachia, cyanobacteria, spirochaetes, greensulfur and green non-sulfur bacteria, Neisseria gonorrhoeae, Neisseriameningitidis, Moraxella catarrhalis, Haemophilus spp such as Haemophilusinfluenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonasaeruginosa, Proteus mirabilis, Enterobacter cloacae, Serratiamarcescens, Helicobacter pylori, and Acinetobacter baumannii.

Apart from the above, it has also been found that the present inventioncan be advantageously used in a composition containing porcine circovirus 2 (PCV2) antigen and optionally Mycoplasma hyopneumoniae antigenand Lawsonia intracellularis antigen for providing a vaccine that afterone single administration provides an adequate immune response againstan infection with porcine circo virus 2 and Mycoplasma hypneumoniae andLawsonia intracellularis

It is noted that the present invention also pertains to an injectablecomposition (in particular a composition having a viscosity less than200 mPa·s, preferably less than 100 mPa·s) comprising the aqueouscomposition referred to here-above.

EMBODIMENTS OF THE INVENTION

In an embodiment the osmolarity of the composition is at least 30%higher than the osmolarity of a 0.9% (w/v) sodium chloride solution(i.e. about 400 mOsmol/l or higher). It has been found that even anosmolarity this high can be used without negative side effects at thesite of administration. This greatly enhances the freedom in use of theacrylic acid polymer. Even amounts significantly over 0.2% as commonlyapplied can now be used while retaining a very viscosity.

In an embodiment the osmolarity of the composition is at least 50%higher than the osmolarity of a 0.9% (w/v) sodium chloride solution(i.e. about 450 mOsmol/l or higher). It has been found that even anosmolarity this high can be used without negative side effects at thesite of administration. This greatly enhances the freedom in use of theacrylic acid polymer. Even amounts significantly over 0.2% as commonlyapplied can now be used while retaining a very viscosity.

In a preferred embodiment the composition comprises more than 0.2% (w/w)of the acrylic acid polymer, or even more than 0.5% (w/w) of the acrylicacid polymer. With these amounts of acrylic acid polymer, thecomposition may be in the form of a viscous gel. By adding the excesselectrolyte to the gelled composition, the viscosity may decrease totypical values between 10-70 mPa·s. The composition will thus be easy toapply using a standard hand-held syringe, and after injection, bydilution of the excess electrolyte, will revert to a highly viscous gel.It has been found that such an acrylic acid polymer gel is particularlysuitable to obtain high antibody titres against antigens which typicallyinduce fever, while decreasing the resultant fever. A composition whichcomprises between 0.8% and 1.6% (w/w) of the acrylic acid polymer isparticularly preferred. In this embodiment, the excess electrolytepreferable provides an osmolarity of typically more than 2-3 times theosmolarity of normal tissue fluid (0.9% sodium chloride solution), thusmore than about 600-900 mOsmol/l. This way, the viscosity beforeadministration can be kept low enough to allow injection. Afterinjection, the electrolyte will dilute and the composition will turninto a gel.

In an embodiment the acrylic acid polymer is a cross linked polymer,preferably using using a polyalkenylether, a polyalcohol and/or adivinylglycol as a cross-linker. Cross-linking the polymer makes thenetwork formation of the polymer chains in the composition less dependedon e.g. temperature and pH, simply because part of the network formationis inherently present in the form of covalent cross-links.

In another embodiment the composition comprises an electrolytecomprising a multicharged cation (that is a cation having a highercharge that 1+, typically 2+ or 3+). It has been found that when using amulticharged cation, the viscosity can be more effectively lowered, evenwhen the osmolarity is the same as when using a monocharged (1+) cation.In a preferred embodiment, the cation is present in a concentration upto 0.05M, preferably up to about 0.03M. The amount of cation that can beused depends on the amount of carboxylate groups in the acrylic acidpolymer solution. Too much cation will induce precipitation. It has beenfound that when using up to these amounts of multicharged cation, theviscosity can be very effectively lowered, while retaining safety uponadministration of the composition.

In an embodiment the biological antigen is an antigen that uponadministration induces fever, i.e. more than 0.5° C., further inparticular more than 1.0° C., more than 1.5° C. or even more than 2.0°C. In practice, many antigens upon administration induce some fever. Themaximum fever that may be induced is often set by regulatoryauthorities. In order to comply with such a maximum, in some cases it isnecessary to lower the amount of antigen to a sub optimal level, therebyaccepting that a non-optimal immunological response is obtained. Forpreventing the actual disease against which the vaccine should provideprotection, this is disadvantageous situation. It has surprisingly beenfound that by using the composition according to the invention, thefever induced by the antigens may be reduced while retaining theimmunological response. Slow-release of the antigens cannot be the solereason for this effect since one would expect that if the fever isreduced due to a slow release, the immunological response would also beless strong. Applicant however found that by using the presentinvention, the fever may be reduced while maintaining an adequateimmunological response.

In an embodiment the antigen is chosen from the group consisting of agram-negative bacterium antigen and a circo virus antigen. In aparticular embodiment the antigen chosen from the group consisting ofActinobacillus pleuropneumoniae antigen, Haemophilus parasuis antigenand porcine circo virus 2 (PCV2) antigen. These antigens areparticularly known for inducing fever upon administration. By using thepresent invention, the fever may be reduced significantly while keepingthe immunological potency of the composition at an adequate level.

The invention will now be further illustrated using the followingexamples.

Example 1 shows the effect of an acrylic acid polymer on viscosity

Example 2 pertains to the physical properties of a composition accordingto the invention, and its safety

Example 3 describes the constitution of compositions according to theinvention

Example 4 shows a vaccination experiment with a composition according tothe invention

Example 5 shows a second vaccination experiment with a compositionaccording to the invention

Example 6 shows a third vaccination experiment with a compositionaccording to the invention

Example 7 describes further compositions according to the invention anda method of formulating these compositions.

EXAMPLE 1

Various aqueous isotonic formulations have been made using across-linked acrylic acid polymer to assess the effect of the polymer onthe viscosity of the composition. The formulations do not compriseantigens (which typically would increase the viscosity). Also, theeffect of the addition of an excess amount of a doubly chargedelectrolyte has been assessed.

All formulations comprised the cross-linked acrylic acid polymerCarbopol 974P (available from BFGoodrich Specialty Chemicals, Cleveland,Ohio). Various amounts of the polymer (0.1 to 1.6% w/w) were solved inan aqueous solution of 0.9% w/v sodium chloride solution. Theviscosities of the formulations were measured with a Brookfieldlaboratory viscometer. The results were as followed:

TABLE 1 Viscosity of various isotonic acrylic acid polymer solutionsCarbopol 974 Viscosity Sample No P w/w mPa · s 1 0.1 3.8 2 0.2 5.0 3 0.570.0 4 0.8 214 5 1.6 4600

It is noted that the aqueous composition of the present invention may beused to make an emulsion with an oil-phase, typically to arrive at awater-in-oil emulsion, an oil-in-water emulsion or awater-in-oil-in-water emulsion. Such an emulsion thus comprises theaqueous composition according to the present invention. Formulating anemulsion will of course impact the viscosity, usually leading to asignificant increase. For example, when an oil (e.g. 10% w/v) isemulsified in an aqueous solution having a viscosity of about 3 mPas,the viscosity may increase to about 25 mPa·s or even higher.

In a next experiment, the effect on viscosity by using an excesselectrolyte was assessed using a 0.8% Carbopol formulation. To thisformulation various amounts of sodium chloride and calcium chloride wereadded to show the effect on viscosity. The results are depicted in Table2.

TABLE 2 Viscosity of various acrylic acid polymer solutions NaClCaCl₂•2H₂O Viscosity Osmolarity Sample No w/v w/v mPa · s mOsmol/l 10.45% 0.16% 748 190 2 0.9% 0.16% 221 340 3 1.5% 0.16% 71 530 4 2.5%0.16% 12 850

As can be understood, the addition of an excess amount of electrolytesignificantly reduces the viscosity of the acrylic acid polymercontaining composition. It was found (see Example 2) that uponinjection, this excess will dilute from the composition into thesurrounding tissue, thereby increasing the viscosity of the compositionto become even a gel. Such a gel will remain at the injection site toawait slow diffusion and degradation of its constituents.

EXAMPLE 2

This example pertains to the physical properties of a compositionaccording to the invention, in particular its gel-formation afterinjection and its release of dispersed content, and its safety uponinjection into animal tissue.

In this experiment four pigs were used. Each pig received 1 ml of acomposition containing 1.6% Carbopol 974P, 2.5% sodium chloride and0.48% calcium chloride (having an approximate osmolarity of 925mOsmol/l) by intramuscular injection. For diagnostic purposes, theformulation additionally comprised 0.075% of the colorant patent blue.Pig number 1 was euthanized 2 hours after injection of the formulationand immediately the muscular tissue was opened to examine the site ofadministration. A clear spot was visible containing a jelly bluesubstance. A spoon was taken and the spot could be removed as a gel. Thegel did not flow. Pig number 2 was euthanized 24 hours afteradministration of the same composition, and its muscular tissue was alsoopened to examine the site of administration. A clear colourless gelcould be spotted at the site of administration, which is an indicationof diffusion of the patent blue into the body of the pig. Pig number 3was euthanized after 48 hours, and pig number 4 after one week. Fromnone of these pigs a gel could be isolated. The blue color wasdisappeared completely.

These results indicate that a gel is present up to 2 days afterinjection. Small molecules diffuse from the gel within 1 day. It isexpected that release of large molecules like proteins is slowed downfor about 2 days in this particular formulation.

The local reactions (i.e. spots comprising deviations in the normaltissue) were also scored at the different time points. The results, sizeof the spots with deviations, are listed below in Table 3. It is notedthat the deviations after 2 hours are mainly restricted to thejelly-spot itself. After 24 hours some deviations in a slightly largertissue volume (5 cm³) were seen, but at a safe (acceptable) level. After1 week, the remaining spot with deviations was virtually gone. The livepigs did not show any signs of local reactions (like stress, itching,redness, biting etc.). Based on these results it can be concluded thatthe composition, despite the fact that the osmolarity is about threetimes the osmolarity of a 0.9% sodium chloride solution, can be regardedas safe.

TABLE 3 Size of the local reaction after IM injection Time after SizeVolume vaccination (hr) (length*width*height in cm) (cm³) 2 4*1*1 4 245*2*0.5 5 48 3*2*0.5 1.5 168 1*0.2*0.3 0.6

EXAMPLE 3

Various compositions were made comprising different biological antigensand different types and amounts of acrylic acid polymers. The amount ofacrylic acid polymer in each case was at least 0.8% w/w to ensure a highviscosity after injection. The general constitution of each compositionis indicated in table 4. It is noted that the indicated viscosity is theviscosity before injection. After injection, the electrolyte will dilutefast from the formulation, whereupon the formulation will become highlyviscous and may remain as a stable gel at the site of administration.

TABLE 4 General constitution of compositions containing biologicalantigens per 1000 grams composition type “0.8” type “1.6” Component(viscosity ± 40 cP) (viscosity ± 70 cP) acrylic acid polymer 8.0 gr 16gr CaCl₂•2H₂O 1.6 gr 4.8 gr  NaCl 9.0 gr 25 gr Antigen x Units x Units(depending on antigen) (depending on antigen) Water added to reach 1000gr added to reach 1000 gr

In general two types of acryl acid polymer comprising formulations weremade, the 0.8 type containing 0.8% acrylic acid polymer and the 1.6 typecontaining 1.6% acrylic acid polymer. In order to keep the viscosity ofthe compositions sufficiently low to enable injecting of the compositionwith a standard hypodermic syringe, electrolyte was added as indicatedhere-above in Table 4. Two different types of cross-linked acrylic acidpolymers were used, viz. Carbopol 974P and Carbopol 971 P (bothobtainable from BFGoodrich). Various types of antigens were being used,derived from the bacteria Actinobacillus pleuropneumoniae, Haemophilisparasuis, Mycoplasma hyopneumoniae and porcine circo virus 2 (PCV2).With these antigens, the following compositions were made:

1 Mhyo/PCV2 Composition

The first composition (denoted “A”) comprised the acrylic acid polymerCarbopol 974P in a concentration of 0.8%. In this compositioninactivated Mycoplasma hyopneumoniae antigens (the same antigens aspresent in the commercially available vaccine Porcilis Mhyo, availablefrom Intervet Schering-Plough Animal Health, Boxmeer, The Netherlands,in the same concentration) and PCV antigens (the same antigens aspresent in the commercially available vaccine Porcilis PCV, availablefrom Intervet Schering-Plough Animal Health, Boxmeer, in the sameconcentration).

The second composition (denoted “B”) comprised the acrylic acid polymerCarbopol 971 P in a concentration of 0.8%. In this compositioninactivated Mycoplasma hyopneumoniae antigens (the same antigens aspresent in the commercially available vaccine Porcilis Mhyo, availablefrom Intervet Schering-Plough Animal Health, Boxmeer, in the sameconcentration) and PCV antigens (the same antigens as present in thecommercially available vaccine Porcilis PCV, available from IntervetSchering-Plough Animal Health, Boxmeer, in the same concentration).

As a first reference composition a composition denoted “C” was madecomprising the same antigens in the same concentration but formulated inthe adjuvant emulsion Emunade, as used in the commercially availablevaccine M+Pac, available from Intervet Schering-Plough Animal Health,Summit, N.J., USA. A strict control formulation “D” was made consistingmerely of PBS solution.

2 APP Composition

A first composition (denoted “E”) comprised the acrylic acid polymerCarbopol 974P in a concentration of 0.8%. This composition comprised thesame Actinobacillus hyopneumoniae antigens (viz. APXI, APX II, APXIIIand OMP), in the same amount, as present in the commercially availablevaccine Porcilis APP, available from Intervet Schering-Plough AnimalHealth, Boxmeer, The Netherlands.

As a reference composition, the commercially available vaccine PorcilisAPP (denoted as “F”) was used.

3 Haemophilus parasuis Composition

A first composition (denoted “G”) comprised the acrylic acid polymerCarbopol 974P in a concentration of 0.8%. This composition comprised thesame Haemophilus parasuis antigens (viz. inactivated cells ofHaemophilus parasuis bacteria), in the same amount, as present in thecommercially available vaccine Porcilis Glässer, available from IntervetSchering-Plough Animal Health, Boxmeer, The Netherlands.

As a reference composition, the commercially available vaccine PorcilisGlässer (denoted as “H”) was used.

The compositions A, B, E and G were made by firstly making a solution ofthe electrolytes in about 70% of the required amount of water. Afterthat the acrylic acid polymer was added and mixed until the polymer iscompletely hydrated. Then the pH of the solution was set to 7.2 usingdrops of a 4M NaOH solution. This product was autoclaved for 20 minutesat 121° C. Then, the product was cooled to approximately 20° C. whilestirring, after which the pH was checked and adjusted if necessary. Thenthe antigens were added while stirring, after which the pH was againchecked and adjusted if necessary. The remaining amount of water wasadded. The product was stirred overnight before filling the containers,and after filling, was stored in a nitrogen gas atmosphere at 2-8° C.

EXAMPLE 4

In the vaccination experiment, four groups of 10 piglets each were used.The compositions A, B, C and D were administered intramuscularly as a 2ml dose at three weeks of age. Rectal temperatures were measured justbefore vaccination (t=0 hr) and 4 hours post vaccination (expectedmaximum temperature rise).

The following results were obtained. No clinical signs of disease whereseen in any of the groups which means that all compositions can beregarded as safe. The average temperatures measured are depicted inTable 5.

TABLE 5 Average temperature after single administration of Mhyp/PCVvaccines T_(av) in ° C. T_(av) in ° C. Composition at t = 0 hr at t = 4hr ΔT in ° C. A 39.8 39.6 −0.2 B 39.8 39.9 +0.1 C 39.6 40.3 +0.7 D(strict control) 39.7 39.5 −0.2

As can be seen, the antigens used may give rise to significanttemperature rise in vaccinated animals (see results with composition C).However, when formulated according to the invention, the temperaturerise may be lower and even completely suppressed.

Next to temperature rise, the antibody titres against the antigens weremeasured, 6 weeks after administration of the composition. The resultsare indicated in Table 6.

TABLE 6 Average titres at 6 weeks after single administration ofMhyp/PCV vaccines Composition Average Mhyo titre Average PCV titre A0.21 10.1 B 0.12 9.2 C 0.06 7.0 D (strict control) 0.02 <3.4

It is noted that composition C is a vaccine that confers protectionagainst Mhyo since the Mhyo-part is the same as the commerciallyavailable single shot vaccine M+Pac. It is thus expected thatcompositions A and B also confer protection against pathogenicMycoplasma hyopneumoniae. With regard to PCV2, it is known that thecommercially available vaccine Porcilis PCV gives rise to protectionwhen the titre is above 9 at about 6 weeks after administration (see i.aEP 2 291 195, results of Example 3).

EXAMPLE 5

Twenty pigs were used at the age of 5½-6 weeks. They were randomlyassigned to two treatment groups of ten pigs each. The pigs receivedcompositions E and F respectively at 6 and 10 weeks of age. The animalswere observed for systemic reactions, in particular rectal temperatureand clinical signs, and local reactions after both vaccinations. Bloodsampling was done at 6, 10, 13 and 23 weeks of age for the determinationof the serological responses to the vaccines. At the age of 23 weeksanimals were post-mortem investigated for local reactions of theinjection sites.

With regard to the latter, at slaughter no unacceptable local reactionswere seen at the injection sites. The average temperature rise wasestablished 4, 6 and 8 hours after administration of the compositions.The results are depicted in Table 7.

TABLE 7 Average temperature rise after administration of APP vaccinesfirst vaccination (prime) second vaccination (boost) CompositionΔT_(av), t = 4 ΔT_(av), t = 6 ΔT_(av), t = 8 ΔT_(av), t = 4 ΔT_(av), t =6 ΔT_(av), t = 8 E 0.8 0.8 0.6 0.5 0.5 0.1 F 0.8 0.7 0.7 0.7 1.0 1.1

As can be seen, the antigens used may give rise to significanttemperature rise in vaccinated animals (see booster results withcomposition F). However, when formulated according to the invention, thetemperature rise may be lower and even completely suppressed (inparticular see results at t=8 hr after booster vaccination).

Next to temperature rise, the antibody titres against the antigens weremeasured, 13 weeks after administration of the composition. The resultsare indicated in Table 8.

TABLE 8 Average titres at 13 weeks after administration of APR vaccinesComposition APXI APXII APXIII OMP E 9.7 11.0 9.7 9.5 F 8.5 10.0 8.5 8.3

Given the fact that the composition according to the invention(composition E) even induces higher titers than the commerciallyavailable and commonly regarded effective vaccine Porcilis APP(composition F), shows that the new composition is an effective vaccineto protect animals against an infection with pathogenic Actinobacilluspleuropneumoniae bacteria.

EXAMPLE 6

In the vaccination experiment, two groups of 6 piglets were used. Thecompositions G and H were administered intramuscularly as a 2 ml dose atone (prime) and four weeks of age (boost). Rectal temperatures weremeasured just before vaccination (t=0 hr) and 6 hours post vaccination(expected maximum temperature rise).

No clinical signs of disease were seen in any of the groups which meansthat all compositions can be regarded as safe. The average temperaturerise (t=6 vs t=0 hr) is depicted in Table 9.

TABLE 9 Average temperature rise after administration of H. parasuisvaccines first vaccination (prime) second vaccination (boost)Composition ΔT_(av), t = 6 hr ΔT_(av), t = 6 hr G 0.6 0.9 H 0.8 1.7

As can be seen, the antigens used may give rise to significanttemperature rise in vaccinated animals (see in particular the boosterresult with composition H). However, when formulated according to theinvention, the temperature rise may be significantly lower.

Next to temperature rise, the antibody titres against the antigens weremeasured, 6 weeks after administration of the composition. The resultsare indicated in Table 10.

TABLE 10 Average titres at 6 weeks after administration of H. parasuisvaccines Composition Average HPS titre G 8.1 H 8.1

Given the fact that the composition according to the invention(composition G) induces the same titer as the commercially available andcommonly regarded effective vaccine Porcilis Glässer (composition H),shows that the new composition is an effective vaccine to protectanimals against an infection with pathogenic Haemophilus parasuisbacteria.

EXAMPLE 7

This example describes further compositions according to the inventionand a method of formulating these compositions. These compositions aredepicted in Table 11. The table indicates units per 1000 ml ofcomposition.

With regard to the antigens mentioned, the Mhyo antigens are the sameantigens as present in the commercially available vaccine Porcilis Mhyo,available from Intervet Schering-Plough Animal Health, Boxmeer, TheNetherlands, in the same concentration per dose (1 U/dose). The PCVantigens are the same antigens as present in the commercially availablevaccine Porcilis PCV, available from Intervet Schering-Plough AnimalHealth, Boxmeer, in the same concentration per dose (5E3 U/dose). TheLawsonia antigens are the numbers of killed whole cells. The resultingvaccine is for use as a one-shot vaccine, 1 ml per dose, to protect pigsagainst an infection with porcine circo virus type 2, Mycoplasmahyopneumoniae and Lawsonia intracellularis. The vaccine is suitable foruse in pigs of an age of three days and older. It is believed that nosite reactions occur, in particular for the formulations A, B and C.

TABLE 11 Compositions comprising PCV, Mhyo and Lawsonia antigens.Component Comp. A Comp. B Comp. C Comp. D units WFI 118.68 237.36 356.04474.72 gram CaCl2 × 2H2O 1.20 2.40 3.60 4.80 gram NaCl 6.25 12.50 18.7525.00 gram Carbopol 4.00 8.00 12.00 16.00 gram 974P NaOH 2.07 4.14 6.218.28 gram Mhyo 1000 1000 1000 1000 U PCV2 5x10E6  5x10E6  5x10E6 5x10E6  U Lawsonia 1x10E11 1x10E11 1x10E11 1x10E11 cells Ad WFI 10071015 1021 1029 grams until Osmolarity 420 550 800 1100 mOs/l

A suitable procedure to constitute the formulations A to D as mentionedin Table 11 is as follows:

-   -   1. Add the WFI to the vessel.    -   2. Make a solution of the sodium chloride and calcium chloride.    -   3. Add the carbopol. Mix the content till a homogeneous        suspension is obtained.    -   4. Adjust the pH of the solution to around 7 (6.9-7.1) with        sodium hydroxide pellets and if necessary re-adjust with        hydrochloric acid (4M).    -   5. Autoclave the product for 20 min at 121° C.    -   6. Cool the product to 20° C. (15-25).    -   7. Check the pH and adjust if necessary.    -   8. Add the antigens while stirring.    -   9. Check the pH and adjust if necessary    -   10. Add the remaining amount of water for injection.    -   11. Store the product at 2-8° C.

1-19. (canceled)
 20. An aqueous composition containing a biologicalantigen and an acrylic acid polymer, wherein the composition comprisesan electrolyte to provide an osmolarity higher than the osmolarity of a0.9% (w/v) sodium chloride solution in water.
 21. The compositionaccording to claim 20, wherein the osmolarity of the composition is atleast 50% higher than the osmolarity of a 0.9% (w/v) sodium chloridesolution.
 22. The composition according to claim 20, wherein thecomposition comprises more than 0.2% (w/w) of the acrylic acid polymer.23. The composition according to claim 22, wherein the compositioncomprises more than 0.5% (w/w) of the acrylic acid polymer.
 24. Thecomposition according to claim 23, wherein the composition comprisesbetween 0.8% and 1.6% (w/w) of the acrylic acid polymer.
 25. Thecomposition according to claim 20, wherein the acrylic acid polymer is across linked polymer.
 26. The composition according to claim 25, whereinthe acrylic acid polymer is cross linked using a polyalkenylether, apolyalcohol and/or a divinylglycol.
 27. The composition according toclaim 20, wherein the composition comprises an electrolyte comprising amulticharged cation.
 28. The composition according to claim 27, whereinthe cation is present in a concentration up to 0.05 M, preferably up to0.03 M.
 29. The composition according to claim 20, wherein thebiological antigen is an antigen that upon administration induces fever.30. The composition according to claim 29, wherein the antigen is chosenfrom the group consisting of a gram-negative bacterium antigen and acirco virus antigen.
 31. The composition according to claim 29, whereinthe antigen is chosen from the group consisting of Actinobacilluspleuropneumoniae antigen, Haemophilus parasuis antigen and porcine circovirus 2 antigen.
 32. The composition according to claim 20, containingporcine circo virus 2 antigen and Mycoplasma hyopneumoniae antigen foruse as a vaccine that after one single administration provides anadequate immune response against an infection with porcine circo virus 2and optionally Mycoplasma hypneumoniae.
 33. An injectable compositioncomprising the aqueous composition according to claim
 20. 34. An acrylicacid polymer for use in an aqueous composition for reducing feverinduced by biological antigens present in the aqueous composition whenthe composition is administered to a subject animal.
 35. The acrylicacid polymer according to claim 34, wherein the composition comprisesmore than 0.2% (w/w), preferably more than 0.5% (w/w) of the acrylicacid polymer.
 36. The acrylic acid polymer according to claim 34,wherein the aqueous solution is hypertonic.